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Yeast Transformation Using Frozen Competent Cells and Single-stranded DNA as a Carrier

Protocol by Katie Kolor based on Schiestl and Gietz (1989) Curr. Genetics 16:339-346; Gietz, et al (1995) Yeast 11:355-360

Preparing competent cells:

1. Inoculate 100 ml yeast synthetic complete medium + 3% glycerol medium from overnight culture. Grow culture to 0.5-1.0 x 107 cells/ml. (2-3x higher efficiency if diluted back to 2 x 106 cells/ml, then grown 2-3 more doublings.) Note: The cells must be grown in glycerol if you are going to freeze them. Glucose-grown cells lose competence upon freezing.
2. Pellet cells gently (~800 x g). Resuspend in 7-8 ml of 1x TE-LiAc.
3. Pellet cells gently. Resuspend in 1 ml of 1x TE-LiAc.
4. Rotate at 23°C for 1 - 1.5 hr.

Cells can be frozen at this point for future use by wrapping the tube in several layers of Kimwipes and/or paper towels and placing them in an ice cream container in a -70oC freezer. It is important to wrap up the tube so that the cells freeze slowly. Thaw cells at room temperature for transformation.

Transformation:

1. Boil 20 µl of herring sperm DNA (8 mg/ml) in an microfuge tube for 15 min. Cool on ice 5 min.
2. Add transforming DNA (up to 5 µg) to the carrier.
3. Add 100 µl of competent cells to the DNA.
4. Add 700 microliters of PEG-TE-LiAc solution (made fresh from the stock solutions). Vortex briefly and gently to mix.
5. Rotate tube at 30°C for 30 min (23°C for ts strains).
6. Transfer tube to 42°C water bath, incubate for 15 min. (Can be reduced to 37°C for ts strains.)
7. Plate an aliquot of transformation mix directly onto selective medium.

Stock solutions

50% Polyethylene glycol 4000
1 M Tris-HCl pH 8.0
1 M Lithium acetate pH 7.5
0.2 M EDTA pH 8.0

1x TE-LiAc

10 mM Tris-HCl pH 8.0
1 mM EDTA
0.1 M Lithium acetate

PEG-TE-LiAc

40% PEG-4000
10 mM Tris-HCl pH 8.0
1 mM EDTA
0.1 M Lithium actetate


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